This renewal application will focus on an observation made in the first 4.5 years of this grant. It was found that gonocytes isolated from mice before or right after birth up until about 3 days after birth could not function efficiently as stem cells in transplant experiments. Cells isolated from animals 4 days plus after birth functioned efficiently as stem cells in these same experiments. The time period for this appearance of transplantation competent cells coincides roughly with the resumption of gonocyte mitosis and the migration of the gonocytes from the interior of the tubule to the basement membrane to form A spermatogonia. Preliminary studies indicate that some of the gonocytes from 3 day old or younger animals migrate to the basement membrane and may even undergo some mitosis but they always fail to initiate spermatogenesis. Even weeks after transplantation a few gonocytes remain on the basement membrane but no colonies of spermatogenic cells are formed and these immature gonocytes fail to function as spermatogenic stem cells. It is our hypothesis that changes in gene expression that are occurring in gonocytes are crucial to the formation of functional (i.e. transplantation competent) germ line stem cells referred to as the "maturation" of gonocytes. The experiments proposed in this application will investigate this hypothesis and attempt to define what changes in gene expression take place during this time in both gonocytes and in testicular somatic cells. The major advantage of our approach is the use of stem cell transplantation as an assay for functional stem cells. Experiments are proposed in 3 Specific aims: 1) Determine if cell culture conditions can transform gonocytes into transplantation competent stem cells. 2) Determine what changes in gene expression occur in the gonocytes and the somatic cells to allow the transformation into transplantation competent stem cells. 3) Test the role of candidate genes in the maturation process using the RNAi system to inhibit specific mRNAs. The proposed studies have significance in obtaining a basic understanding of what is required to form a spermatogenic stem cell and in gaining more practical insight into what manipulations could possibly be performed on primordial germ cells and gonocytes to create a source of cells for transplantation. This research could have implications in approaching human infertility problems where the maturation of primordial germ cells or gonocytes into functional stem cells is aberrant.